Personalized medicine is the application of genomic and molecular data to better target the delivery of health care to specific patients, facilitate the discovery and clinical testing of new products, and help determine a person's predisposition to a particular disease or condition.
On a technical level, personalized medicine depends on the identification and detection of proteins, genes and genetic variation (“biomarkers”) that play a role in a given disease. Rodland, Clin Biochem. 2004 July; 37(7):579-83. The presence or absence of certain biomarkers is then correlated with the incidence of a particular disease or disease predisposition. However, currently available methods for biomarker analysis are associated with long waiting periods, high cost and numerous technical hurdles.
Mass spectrometry (MS) is an important method for the characterization of proteins in biological samples. MS involves ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass or mass-to-charge ratios. In a typical MS procedure, a sample is loaded onto the MS instrument, and undergoes vaporization. The components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of positively charged particles (ions). The positive ions are then accelerated by a magnetic field. The computation of the mass-to-charge ratio of the particles is based on the details of motion of the ions as they transit through electromagnetic fields, and detection of the ions.
The two primary methods for MS ionization are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). Generally, however, proteins must be enzymatically digested into smaller peptides using a protease prior to ionization and analysis. The resulting peptides are introduced into the mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. Identification of certain peptides allows the technician to infer the existence of particular proteins in an original sample.
Proteins of interest to biological researchers are usually part of a very complex mixture of other proteins and molecules that co-exist in the biological medium (e.g., serum, blood, urine, tissue sample, mucous, saliva, stool, etc.). This presents two significant problems. First, the two ionization techniques used for large molecules (i.e., ESI and MALDI) only work well when the mixture contains roughly equal amounts of constituents. However, different proteins tend to be present in widely differing amounts in biological samples. If such a mixture is ionized using ESI or MALDI, the more abundant species have a tendency to “drown” or suppress signals from less abundant ones. The second problem is that the mass spectrum from a complex mixture is very difficult to interpret because of the overwhelming number of mixture proteins. This complexity is exacerbated by the usually necessary enzymatic digestion of a protein prior to MS analysis. As such, biological media must generally be laboriously pre-processed before MS analysis can be performed.
Two pre-processing methods are usually used to fractionate proteins, or their peptide products from an enzymatic digestion, prior to MS analysis: Two-dimensional (“2-D”) gel electrophoresis and high performance liquid chromatography (“HPLC”).
Two-dimensional (“2-D”) gel electrophoresis separates a mixture of proteins by two properties in two dimensions on 2D gels. See e.g., Kossowska et al., Postepy Hig Med Dosw (Online). 2009 Nov. 12; 63:549-63; Ma et al., Electrophoresis. 2009 August; 30(15):2591-9.
2-D electrophoresis begins with 1-D electrophoresis but then separates the molecules by a second property in direction 90 degrees from the first. The result is that the molecules are spread out across a 2-D gel. Because it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis. However, 2-D electrophoresis is a time (30 minutes to 12 hours per sample) and labor intensive process requiring complex as well as very expensive equipment and highly trained and experienced technicians.
Fractionation of peptides after enzymatic digestion into multiple fractions by HPLC (e.g. by SCX) prior to MS analysis is also commonly used. Yet all these fractions must be analyzed separately, greatly increasing time and effort spent on a single sample.
Apart from these fractionation methods prior to MS analysis, MS itself is often combined with HPLC which is generally referred to as “LC-MS.” LC-MS is a powerful technique used for many applications which has very high sensitivity and specificity.
The “bottom-up” approach to proteomics involves protease digestion and denaturation followed by LC-MS with peptide mass fingerprinting; or LC-MS/MS (or “two-stage” or “tandem MS”) to derive the sequence of individual peptides. See e.g., Shi et al., Anal Chem. 2009, Nov. 19; Grimsrud et al., Plant Physiol. 2009 Nov. 18; Kesic et al., Retrovirology. 2009 Nov. 17; 6:105; Kilpatrick et al., Anal Chem. 2009 Oct. 15; 81(20):8610-6. Wu et al., Anal Biochem. 2009 Nov. 3; Hartwig et al., Arch Physiol Biochem. 2009 Nov. 4; Caron et al., Mol Cell Proteomics. 2007 July; 6(7):1115-22. Epub 2007 Mar. 20.
A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation. Tandem mass spectrometry allow for a variety of experimental sequences. Normally, a tandem MS has at least two mass spectrometers in series connected by a chamber that can break a molecule into pieces. A sample peptide is sorted and weighed in the first mass spectrometer (MS1), broken into pieces in the collision cell, and a peptide piece or pieces again sorted and weighed in the second mass spectrometer (MS2). Many commercial mass spectrometers are designed to expedite the execution of sequences as single reaction monitoring (SRM), multiple reaction monitoring (MRM), and precursor ion scan. In SRM, the MS1 allows only a single mass through and MS2 monitors for a single user defined fragment ion. MRM allows for multiple user defined fragment ions. Unfortunately, SRM and MRM require complex and expensive instrumentation and computer equipment.
LC-MS/MS is most commonly used for proteomic analysis of complex samples. Samples of complex biological fluids like human serum may be run through an LC-MS/MS system and result in over 1000 proteins being identified. However, to achieve such results, samples are generally first separated on an SDS-PAGE gel or strong cation exchange (“SCX”) HPLC.
Recently, it has been reported that the MRM assay can be coupled with a strategy to enrich certain peptides: Stable Isotope Standards with Capture by Anti-Peptide Antibodies (“SISCAPA”). In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, SRM/MRM tandem MS is used to quantify the peptides (natural and labeled). See Whiteaker et al., Mol Cell Proteomics. 2009 Oct. 20; Ahn et al., J Proteome Res. 2009 September; 8(9):4216-24; Kuhn et al., Clin Chem. 2009 June; 55(6):1108-17. Epub 2009 Apr. 16; Anderson et al., Mol Cell Proteomics. 2009 May; 8(5):995-1005. Epub 2009 Feb. 4; Anderson et al., Mol Cell Proteomics. 2006 April; 5(4):573-88. Epub 2005 Dec. 6; Anderson et al., J Proteome Res. 2004 March-April; 3(2):235-44; US Pub. Nos. 20060154318, 20070231909, 20080217254.
The inventor has developed a simplified method that can rapidly fractionate and analyze a biological sample for the presence or absence of a particular protein or peptide analytes by measured accurate mass alone (i.e., using only M1) without the need to for complex and expensive tandem MS or MS2 procedures and equipment.